As precartilage mesenchymal cells differentiate into cartilage cells they stop producing the extracellular matrix glycoproteins type I collagen and fibronectin and begin producing the matrix glycoprotein type II collagen. Using a virtually pure population of chick limb nonmyogenic precartilage cells we have previously identified, definitive chick cartilage cells, and cloned cDNA probes for the fibronectin (FN), Alpha2(I, and Alpha1(II) collagen genes, we propose to investigate changes in the domain organization of chromatin during chondrogenesis in order to elucidate mechanisms of changing gene expression during development. We have previously determined that a developmentally-regulated, DNA binding, precartilage chromatin protein, PCP 35.5 is localized near DNAase I-sensitive regions of precartilage chromatin and is progressively modified from a basic to acidic form during differentiation by a process apparently dependent on cyclic AMP. We therefore propose to determine the DNAase I sensitivities of the FN, Alpha2(I), and AlphaI(II) genes in precartilage and cartilage nuclei, the presence or absence of these genes in the DNAase I sensitive sequences near PCP 35.5, and possible changes in the domain organization of these genes in nuclei whose PCP 35.5 has been caused to undergo cyclic AMP dependent phosphorylation in vitro. These studies should help define the role of modifications of PCP 35.5 in the reorganization of chromatin during chondrogenesis. Finally, we propose to hybridize the cDNA probes for the FN, Alpha 2 (I) and Alpha 1 (II) collagen genes to filter transfers of electrophoresed nuclear and cytoplasmic RNA to determine actual levels of expression of these genes in the two cell types, and to relate these findings to those on the domain reorganization of chromatin.